CN106124777A - Acridine labelling conjugate and preparation method thereof, chemical luminescence reagent kit - Google Patents
Acridine labelling conjugate and preparation method thereof, chemical luminescence reagent kit Download PDFInfo
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Abstract
The invention discloses acridine labelling conjugate and preparation method thereof, chemical luminescence reagent kit, acridine labelling conjugate includes acridine substituent, carrier protein and the albumen to be marked being sequentially connected with；Described carrier protein is containing carboxyl and the albumen of amino, modified protein, polypeptide or modified polypeptides；Described albumen to be marked is the albumen containing amino, modified protein, polypeptide or modified polypeptides.The carrier protein of this acridine labelling conjugate reacts formation chemical bond by the amino on carrier protein and is connected with acridine substituent, amino on albumen to be marked forms NH CO structure with the carboxyl reaction on carrier protein thus carrier protein and albumen to be marked is connected together, binding site determines relatively, avoiding the avtive spot formation interference that acridine substituent is treated on labelled protein, the activity of this acridine labelling conjugate is of a relatively high.
Description
Technical field
The present invention relates to vitro detection field, particularly relate to a kind of acridine labelling conjugate and preparation method thereof, chemistry
Light test kit.
Background technology
Chemiluminescent labeling immunoassay, also known as chemiluminescence immune assay (CLIA), is with the direct labelling of chemiluminescence agent
The immune analysis method of antigen, hapten or antibody.Chemiluminescent substance for labelling includes acridine substituent, according to replacement
The difference of base, acridine substituent is divided into two classes: acridinium ester (acridinium ester, AE) and acridine sulfonamide, both at
Effective luminous marker, by starting luminescence reagent (NaOH, H2O2) effect and luminous, strong directly luminescence was at one second
Inside complete, for quick flashing.
Acridine substituent as chemiluminescent labels for immunoassay, its chemical reaction is simple, quickly, need not be catalyzed
Agent, detection small molecule antigens uses competition law, and macromole antigen then uses sandwich assay, and non-specific binding is few, and background is low, and greatly
The combination of molecule will not reduce produced light quantity, thus increases sensitivity.This compounds is feature for luminous mechanism
It is: 1, in luminescence-producing reaction before forming excited electronic state intermediate, the non-luminous substituent group part being linked on acridine ring
Departing from from acridine ring and come, the most non-luminous component separates with luminous component, thus its luminous efficiency is the most by replacing base junction
The impact of structure.2, acridinium ester or acridine sulfamide compound chemiluminescence need not catalyst, having H2O2Dilute alkaline solution
In can be luminous.Therefore being applied to chemiluminescence detection tool have many advantages, advantage mainly has: 1. background luminescence is low, noise
Ratio is high；2. luminescence-producing reaction interference factor is few；3. light release is quickly concentrated, luminous efficiency is high, luminous intensity is big；4. it is prone to and albumen
After matter connection and connection, photon productivity does not reduces；5. label is stable (can preserve the several months long at 2-8 DEG C).Therefore acridine takes
It is class chemiluminescent labels highly effective, extraordinary for thing.
Acridine labelling conjugate is acridine substituent and thing to be marked (antibody, antigen, etc.) combines the complex obtained.A word used for translation
The quality of pyridine labelling conjugate quality is directly connected to the success or not of chemiluminescence immunoassay technology, is therefore referred to as key
Reagent.
The preparation method of the most conventional acridine labelling conjugate is carbodiimide cross-linking method, with carbodiimide cross-linking agent is
Bridge, makes acridine substituent and protein binding to be marked.But, in the acridine labelling conjugate that traditional method prepares, acridine replaces
Thing is combined by carbodiimide with albumen to be marked, and acridine substituent is often treated the avtive spot on labelled protein and formed dry
Disturb, cause the activity reduction of acridine labelling conjugate, affect the sensitivity of immunoassay.
Summary of the invention
Based on this, it is necessary to provide of a relatively high acridine labelling conjugate of a kind of activity and preparation method thereof, chemistry to send out
Light test kit.
A kind of acridine labelling conjugate, including the acridine substituent being sequentially connected with, carrier protein and albumen to be marked；
Described carrier protein is containing carboxyl and the albumen of amino, modified protein, polypeptide or modified polypeptides, described carrier egg
The white amino passed through on described carrier protein reacts formation chemical bond and is connected with described acridine substituent；
Described albumen to be marked is the albumen containing amino, modified protein, polypeptide or modified polypeptides, described albumen to be marked
On amino with on described carrier protein carboxyl reaction formed-NH-CO-structure thus described carrier protein and described waiting are marked
Note albumen connects together.
In one embodiment, described acridine substituent is acridinium ester, acridinic acid, acridinium carboxamide or acridine sulfonamide.
In one embodiment, described carrier protein is bovine serum albumin, chicken serum albumin or hemocyanin.
In one embodiment, described albumen to be marked is antigen, hapten or antibody.
The preparation method of a kind of above-mentioned acridine labelling conjugate, comprises the steps:
Combine with obtaining acridine substituent-carrier protein after acridine substituent and carrier protein covalent cross-linking, fully reaction
Thing, wherein, described carrier protein is containing carboxyl and the albumen of amino, modified protein, polypeptide or modified polypeptides, described carrier egg
The white amino passed through on described carrier protein reacts formation chemical bond and is connected with described acridine substituent；
Described acridine substituent-carrier protein conjugate is purified；
With cross-linking agent, the carboxyl on described acridine substituent-carrier protein conjugate after purification is activated；And
With the described acridine substituent-carrier protein conjugate after activated carboxylic and protein-crosslinking to be marked, fully react
After obtain acridine labelling conjugate, wherein, described acridine labelling conjugate includes acridine substituent, the carrier protein being sequentially connected with
With albumen to be marked, described albumen to be marked is the albumen containing amino, modified protein, polypeptide or modified polypeptides, described in wait to mark
Amino on note albumen forms-NH-CO-structure thus by described carrier protein and institute with the carboxyl reaction on described carrier protein
State albumen to be marked to connect together.
In one embodiment, in the operation of described acridine substituent and carrier protein covalent cross-linking, described acridine takes
Mol ratio for thing and described carrier protein is 100～20000:1.
In one embodiment, the described acridine substituent-carrier protein conjugate after described activated carboxylic with wait to mark
In the operation of note protein-crosslinking, described acridine substituent-carrier protein conjugate is 5:1 with the mol ratio of described albumen to be marked
～1:5.
In one embodiment, described cross-linking agent is on described acridine substituent-carrier protein conjugate after purification
Carboxyl carry out in the operation activated, described cross-linking agent includes carbodiimide and N-Hydroxysuccinimide.
In one embodiment, described carbodiimide is selected from dicyclohexylcarbodiimide, 1-(3-dimethylamino-propyl)-3-
Ethyl carbodiimide and N, at least one in N'-DIC, described carbodiimide and described acridine substituent-
The mol ratio of carrier protein conjugate is 10～5000:1；
Described N-Hydroxysuccinimide is selected from N-hydroxy-succinamide and NHS at least
One, described carbodiimide is 5:1～1:10 with the mol ratio of described N-Hydroxysuccinimide.
A kind of chemical luminescence reagent kit, forms above-mentioned acridine labelling conjugate, describedization for combining albumen to be marked
Learn luminescence reagent box to include: acridine substituent and carrier protein；
Described carrier protein is containing carboxyl and the albumen of amino, modified protein, polypeptide or modified polypeptides, described carrier egg
Formation chemical bond can be reacted with described acridine substituent by the amino on described carrier protein in vain to be connected；
Described albumen to be marked is the albumen containing amino, modified protein, polypeptide or modified polypeptides, described albumen to be marked
On amino can form-NH-CO-structure with the carboxyl reaction on described carrier protein thus by described carrier protein and described
Albumen to be marked connects together.
This acridine labelling conjugate includes acridine substituent, carrier protein and albumen to be marked, the carrier being sequentially connected with
Albumen reacts formation chemical bond by the amino on carrier protein and is connected with acridine substituent, the amino on albumen to be marked and load
Carboxyl reaction on body protein forms-NH-CO-structure thus is connected together with albumen to be marked by carrier protein, bound site
Point determines relatively, it is to avoid acridine substituent is treated the avtive spot on labelled protein and formed interference, and this acridine labelling combines
The activity of thing is of a relatively high.Additionally, carrier protein can make the sterically hindered increase of acridine labelling conjugate, thus add acridine
The use sensitivity of labelling conjugate.
Accompanying drawing explanation
Fig. 1 is the flow chart of the preparation method of the acridine labelling conjugate of an embodiment；
Fig. 2 is the series concentration TSH antigen Sample result scatterplot obtained in test case；
Fig. 3 is the series concentration E2 antigen Sample result scatterplot obtained in test case.
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, real with concrete below in conjunction with the accompanying drawings
Execute example the detailed description of the invention of the present invention is described in detail.Elaborate a lot of detail in the following description so that
Fully understand the present invention.But the present invention can implement to be much different from alternate manner described here, art technology
Personnel can do similar improvement in the case of intension of the present invention, and therefore the present invention is not embodied as by following public
Restriction.
A kind of acridine labelling conjugate, including the acridine substituent being sequentially connected with, carrier protein and albumen to be marked.
Carrier protein is that carrier protein is by carrying containing carboxyl and the albumen of amino, modified protein, polypeptide or modified polypeptides
Amino on body protein reacts formation chemical bond and is connected with acridine substituent.
Carrier protein can be inherently to have carboxyl and the albumen of amino or polypeptide, it is also possible to for by introducing after modifying
Carboxyl and the modified protein of amino or modified polypeptides.
In present embodiment, carrier protein can be bovine serum albumin, chicken serum albumin or hemocyanin.
Albumen to be marked is the albumen containing amino, modified protein, polypeptide or modified polypeptides, the amino on albumen to be marked
Form-NH-CO-structure with the carboxyl reaction on carrier protein thus carrier protein and albumen to be marked are connected together.
Albumen to be marked can be albumen or the polypeptide inherently with amino, it is also possible to for by introducing ammonia after modifying
The modified protein of base or modified polypeptides.
In present embodiment, albumen to be marked is antigen, hapten or antibody.
Acridine substituent can be acridinium ester (DMAE-NHS, AE-NHS), acridinic acid (9-acridine formic acid), acridinium carboxamide or
Acridine sulfonamide (NSP-SA-NHS).According to the concrete difference of acridine substituent, the amino on carrier protein and acridine substituent
On different groups (carboxyl, succinimide ester etc.) reaction formed chemical bond connect.
Concrete, acridinium ester can be AE-NHS (10-methylacridinium-9-N-succinimide ester formic acid esters), DMAE-
NHS (2 ', 6 '-dimethyl-4 '-(N-succinimidyloxycarbonyl) phenyl-acridine-9-formic acid esters), ME-DMAE-NHS (2 ',
6 '-dimethyl-carbonyl phenyl-10-methyl-9-acridine formic acid esters-4 '-N-succinimide ester-trifluoromethyl sulfonic acid) etc..
As a example by acridinium ester is as AE-NHS, now, acridine labelling conjugate has a following structural formula:
Wherein,For carrier protein, with amino residue and residual carboxylic acid group on carrier protein,For treating
Labelled protein, with amino residue on albumen to be marked.
This acridine labelling conjugate includes acridine substituent, carrier protein and albumen to be marked, the carrier being sequentially connected with
Albumen reacts formation chemical bond by the amino on carrier protein and is connected with acridine substituent, the amino on albumen to be marked and load
Carboxyl reaction on body protein forms-NH-CO-structure thus is connected together with albumen to be marked by carrier protein, bound site
Point determines relatively, it is to avoid acridine substituent is treated the avtive spot on labelled protein and formed interference, and this acridine labelling combines
The activity of thing is of a relatively high.Additionally, carrier protein can make the sterically hindered increase of acridine labelling conjugate, thus add acridine
The use sensitivity of labelling conjugate.
Additionally, carrier protein can make the sterically hindered increase of acridine labelling conjugate, thus add acridine labelling and combine
The use sensitivity of thing.
Compared with conventional art, in this acridine labelling conjugate containing carrier protein, the active sites of albumen to be marked
Point is effectively protected, it is to avoid protein inactivation to be marked, by chemical bond by acridine substituent, carrier protein and albumen to be marked
It is sequentially connected with, there is the features such as stable, connection amount is controlled.
This acridine labelling conjugate is used directly for detection and the quantitative analysis of chemiluminescence immune assay, and energy
Solve the shortcomings such as acridine labelling conjugate raw material inactivation, signal difference prepared by the methods such as traditional carbodiimide cross-linking method.
The preparation method of above-mentioned acridine labelling conjugate as shown in Figure 1, comprises the steps:
Acridine substituent-carrier protein is obtained after S10, use acridine substituent and carrier protein covalent cross-linking, fully reaction
Conjugate.
In operation with acridine substituent and carrier protein covalent cross-linking, the mol ratio of acridine substituent and carrier protein is
100～20000:1.
Preferably, the mol ratio of acridine substituent and carrier protein is 500～5000:1.
Carrier protein is that carrier protein is by carrying containing carboxyl and the albumen of amino, modified protein, polypeptide or modified polypeptides
Amino on body protein reacts formation chemical bond and is connected with acridine substituent.
Carrier protein can be inherently to have carboxyl and the albumen of amino or polypeptide, it is also possible to for by introducing after modifying
Carboxyl and the modified protein of amino or modified polypeptides.
In present embodiment, carrier protein can be bovine serum albumin, chicken serum albumin or hemocyanin.
Acridine substituent can be acridinium ester (DMAE-NHS, AE-NHS), acridinic acid (9-acridine formic acid), acridinium carboxamide or
Acridine sulfonamide (NSP-SA-NHS).According to the concrete difference of acridine substituent, the amino on carrier protein and acridine substituent
On different groups (carboxyl, succinimide ester etc.) reaction formed chemical bond connect.
Concrete, acridinium ester can be AE-NHS (10-methylacridinium-9-N-succinimide ester formic acid esters), DMAE-
NHS (2 ', 6 '-dimethyl-4 '-(N-succinimidyloxycarbonyl) phenyl-acridine-9-formic acid esters), ME-DMAE-NHS (2 ',
6 '-dimethyl-carbonyl phenyl-10-methyl-9-acridine formic acid esters-4 '-N-succinimide ester-trifluoromethyl sulfonic acid) etc..
As a example by acridinium ester is as AE-NHS, acridine substituent and carrier protein covalent cross-linking obtain acridine substituent-carrier
The reaction equation of protein conjugates is as follows:
Wherein,For carrier protein, with amino and carboxyl on carrier protein, AE-NHS passes through with carrier protein
Amido link connects.
The concrete course of reaction of above-mentioned reaction equation is: add carrier protein in buffer, adds excess after fully dissolving
Acridinium ester, in 4 DEG C～37 DEG C react 0.5h～12h, obtain acridinium ester-carrier protein conjugate through purification after reaction.Its
In, buffer is phosphate buffer, carbonate buffer solution, 2-(N-morpholino) ethyl sulfonic acid (MES) buffer or piperazine-N, N '
Double (2-ethanesulfonic acid) (PIPES) buffer, the pH of course of reaction is 4～10.
Owing to acridinium ester is excessive relative to carrier protein, therefore the amino on carrier protein can be considered as having reacted
Entirely, will not be at war with the amino on albumen to be marked in next step, such that it is able to acridinium ester-carrier protein knot need not be closed
Amino on compound.
S20, the acridine substituent-carrier protein conjugate obtaining S10 are purified.
The operation that acridine substituent-carrier protein conjugate is purified can be ultrafiltration purification, desalting column purification and
One or more combinations in dialysis several operation of purification.
Carboxyl on S30, the substituent of the acridine after purification-carrier protein conjugate obtained S20 with cross-linking agent is carried out
Activation.
Cross-linking agent includes that carbodiimide and N-Hydroxysuccinimide, carbodiimide are combined with acridine substituent-carrier protein
The mol ratio of thing is 10～5000:1, and carbodiimide is 5:1～1:10 with the mol ratio of N-Hydroxysuccinimide.
Preferably, carbodiimide is selected from dicyclohexylcarbodiimide, 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide
And at least one in N, N'-DIC.
Preferably, carbodiimide is 50～1000:1 with the mol ratio of acridine substituent-carrier protein conjugate.
Preferably, during N-Hydroxysuccinimide is selected from N-hydroxy-succinamide and NHS extremely
Few one.
Preferably, carbodiimide is 2:1～1:5 with the mol ratio of N-Hydroxysuccinimide.
Preferably, after the carboxyl that S30 is additionally included on acridine substituent-carrier protein conjugate is activated, add sulfydryl
Ethanol cancellation cross linker active (or purified removing cross-linking agent), obtains the substituent of the acridine after activated carboxylic-carrier protein
Amino in acridine substituent-carrier protein conjugate after conjugate, and this activated carboxylic is closed.
Acridine substituent-carrier protein conjugate after S40, the activated carboxylic obtained with S30 and protein-crosslinking to be marked,
Fully obtain acridine labelling conjugate after reaction.
The acridine labelling conjugate obtained includes acridine substituent, carrier protein and the albumen to be marked being sequentially connected with.
Albumen to be marked is the albumen containing amino, modified protein, polypeptide or modified polypeptides, the amino on albumen to be marked
Form-NH-CO-structure with the carboxyl reaction on carrier protein thus carrier protein and albumen to be marked are connected together.
Albumen to be marked can be albumen or the polypeptide inherently with amino, it is also possible to for by introducing amino after modifying
Modified protein or modified polypeptides.
In present embodiment, albumen to be marked is antigen, hapten or antibody.
In operation with the acridine substituent-carrier protein conjugate after activated carboxylic and protein-crosslinking to be marked, acridine
Substituent-carrier protein conjugate is 5:1～1:5 with the mol ratio of albumen to be marked.
Preferably, acridine substituent-carrier protein conjugate is 2:1～1:2 with the mol ratio of albumen to be marked.
As a example by acridinium ester is as AE-NHS, use carbodiimide and N-Hydroxysuccinimide to acridine substituent-carrier egg
The carboxyl of white conjugate activates, the acridine substituent-carrier protein conjugate after activated carboxylic and protein-crosslinking to be marked,
The reaction equation obtaining acridine labelling conjugate is as follows:
Wherein,For carrier protein, with amino residue and residual carboxylic acid group on carrier protein,For treating
Labelled protein, with amino residue on albumen to be marked.
The concrete course of reaction of above-mentioned reaction equation is: adds acridine substituent-carrier protein conjugate in buffer, fills
Divide after dissolving, add EDC and NHS, react 10min in 25 DEG C, be subsequently adding albumen to be marked, react 0.5h in 4 DEG C～37 DEG C
～12h, obtain acridine labelling conjugate through purification after reaction.Wherein, buffer is phosphate buffer, carbonate buffer
Liquid, 2-(N-morpholino) ethyl sulfonic acid (MES) buffer or piperazine-N, N ' double (2-ethanesulfonic acid) (PIPES) buffer, reacted
The pH of journey is 4～10.
The preparation method of this acridine labelling conjugate, carrier protein is by the amino on carrier protein and acridine substituent
Reaction forms chemical bond and connects, the carboxyl reaction on the amino on albumen to be marked and carrier protein formed-NH-CO-structure from
And carrier protein is connected together with albumen to be marked, binding site determines relatively, and binding site determines relatively, it is to avoid a word used for translation
Pyridine substituent is treated the avtive spot on labelled protein and is formed interference, a word used for translation that the preparation method of this acridine labelling conjugate prepares
The activity of pyridine labelling conjugate is of a relatively high.
Additionally, carrier protein can make the sterically hindered increase of acridine labelling conjugate, thus add acridine labelling and combine
The use sensitivity of thing.
The invention also discloses a kind of chemical luminescence reagent kit, form above-mentioned acridine labelling for combining albumen to be marked
Conjugate.
Chemical luminescence reagent kit includes: acridine substituent and carrier protein.
Acridine substituent can be acridinium ester (DMAE-NHS, AE-NHS), acridinic acid (9-acridine formic acid), acridinium carboxamide or
Acridine sulfonamide (NSP-SA-NHS).According to the concrete difference of acridine substituent, the amino on carrier protein and acridine substituent
On different groups (carboxyl, succinimide ester etc.) reaction formed chemical bond connect.
Concrete, acridinium ester can be AE-NHS (10-methylacridinium-9-N-succinimide ester formic acid esters), DMAE-
NHS (2 ', 6 '-dimethyl-4 '-(N-succinimidyloxycarbonyl) phenyl-acridine-9-formic acid esters), ME-DMAE-NHS (2 ',
6 '-dimethyl-carbonyl phenyl-10-methyl-9-acridine formic acid esters-4 '-N-succinimide ester-trifluoromethyl sulfonic acid) etc..
Carrier protein is containing carboxyl and the albumen of amino, modified protein, polypeptide or modified polypeptides, and carrier protein can lead to
Cross the amino on carrier protein react with acridine substituent formation chemical bond be connected.
Carrier protein can be inherently to have carboxyl and the albumen of amino or polypeptide, it is also possible to for by introducing after modifying
Carboxyl and the modified protein of amino or modified polypeptides.
In present embodiment, carrier protein can be bovine serum albumin, chicken serum albumin or hemocyanin.
Albumen to be marked is the albumen containing amino, modified protein, polypeptide or modified polypeptides, the amino on albumen to be marked
Form-NH-CO-structure with the carboxyl reaction on carrier protein thus carrier protein and albumen to be marked are connected together.
Albumen to be marked can be albumen or the polypeptide inherently with amino, it is also possible to for by introducing ammonia after modifying
The modified protein of base or modified polypeptides.
In present embodiment, albumen to be marked is antigen, hapten or antibody.
Preferably, at least one during this chemical luminescence reagent kit also includes centrifugal desalting column and centrifugal ultrafiltration pipe.
This chemical luminescence reagent kit can sequentially form chemistry by acridine substituent, carrier protein and albumen to be marked
Bonded, binding site determines relatively, it is to avoid acridine substituent is treated the avtive spot on labelled protein and formed interference, is formed
The activity of acridine labelling conjugate of a relatively high.
Additionally, carrier protein can make the sterically hindered increase of acridine labelling conjugate, thus add acridine labelling and combine
The use sensitivity of thing.
It it is below specific embodiment.
Embodiment 1
Being dissolved in by 1mg BSA in 1mL150mM PBS (pH 7.4), (10mg/mL is molten to add 40 μ L acridinium esters
Solution is in DMF) react 4h in 25 DEG C, with the desalting column (Thermofish company) of 5mL 7KD molecular cut off with 150mM PBS
(pH 7.4) buffer, as changing liquid buffer, crosses post 3 times, removes free acridinium ester and byproduct of reaction, obtain acridine-
BSA solution.
EDC (final concentration of 10mmol/L) and NHS is added (final concentration of in the acridine-BSA solution that above-mentioned purification is crossed
20mmol/L).After 25 DEG C are reacted 10 minutes, add TSH antibody (producer: Santa cruz biotechnology, article No.: sc-
418393), mixing, be placed in 25 DEG C reaction 4h, with the desalting column (Thermofish company) of 5mL 7KD molecular cut off with
150mM PBS (pH 7.4) buffer, as changing liquid buffer, crosses post 3 times, removes free EDC, NHS and byproduct of reaction,
Obtain the TSH monoclonal antibody solution of the band BSA of acridinium ester label.
Embodiment 2
Being dissolved in by 1mg BSA in 1mL 150mM PBS (pH 7.4), (10mg/mL is molten to add 40 μ L acridinium esters
Solution is in DMF) react 4h in 25 DEG C, with the desalting column (Thermofish company) of 5mL 7KD molecular cut off with 150mM PBS
(pH 7.4) buffer, as changing liquid buffer, crosses post 3 times, removes free acridinium ester and byproduct of reaction, obtain acridine-
BSA solution.
EDC (final concentration of 10mmol/L) and NHS is added (final concentration of in the acridine-BSA solution that above-mentioned purification is crossed
20mmol/L).After 25 DEG C are reacted 10 minutes, add estradiol antigen (producer: abcam, article No.: ab120657), mixing, be placed in
25 DEG C of reaction 4h, buffer with 150mM PBS (pH 7.4) with the desalting column (Thermofish company) of 5mL 7KD molecular cut off
Liquid, as changing liquid buffer, crosses post 3 times, removes free EDC, NHS and byproduct of reaction, obtains the band BSA of acridinium ester label
Estradiol antigenic solution.
Embodiment 3
Being dissolved in by 1mg OVA in 1mL 150mM PBS (pH 7.4), (10mg/mL is molten to add 60 μ L acridinium esters
Solution is in DMF) react 4h in 25 DEG C, with the desalting column (Thermofish company) of 5mL 7KD molecular cut off with 150mM PBS
(pH 7.4) buffer, as changing liquid buffer, crosses post 3 times, removes free acridinium ester and byproduct of reaction, obtain acridine-
OVA solution.
EDC (final concentration of 10mmol/L) and NHS is added (final concentration of in the acridine-OVA solution that above-mentioned purification is crossed
20mmol/L).After 25 DEG C are reacted 10 minutes, add TSH antibody (producer: Santa cruz biotechnology, article No.: sc-
418393), mixing, be placed in 25 DEG C reaction 4h, with the desalting column (Thermofish company) of 5mL 7KD molecular cut off with
150mM PBS (pH 7.4) buffer, as changing liquid buffer, crosses post 3 times, removes free EDC, NHS and byproduct of reaction,
Obtain the TSH monoclonal antibody solution of the band OVA of acridinium ester label.
Embodiment 4
Being dissolved in by 1mg OVA in 1mL 150mM PBS (pH 7.4), (10mg/mL is molten to add 60 μ L acridinium esters
Solution is in DMF) react 4h in 25 DEG C, with the desalting column (Thermofish company) of 5mL 7KD molecular cut off with 150mM PBS
(pH 7.4) buffer, as changing liquid buffer, crosses post 3 times, removes free acridinium ester and byproduct of reaction, obtain acridine-
OVA solution.
EDC (final concentration of 10mmol/L) and NHS is added (final concentration of in the acridine-OVA solution that above-mentioned purification is crossed
20mmol/L).After 25 DEG C are reacted 10 minutes, add estradiol antigen (producer: abcam, article No.: ab120657), mixing, be placed in
25 DEG C of reaction 4h, buffer with 150mM PBS (pH 7.4) with the desalting column (Thermofish company) of 5mL 7KD molecular cut off
Liquid, as changing liquid buffer, crosses post 3 times, removes free EDC, NHS and byproduct of reaction, obtains the band OVA of acridinium ester label
Estradiol antigenic solution.
Embodiment 5
Being dissolved in by 1mg KLH in 1mL 150mM PBS (pH 7.4), (10mg/mL is molten to add 5 μ L acridinium esters
Solution is in DMF) react 4h in 25 DEG C, with the desalting column (Thermofish company) of 5mL 7KD molecular cut off with 150mM PBS
(pH 7.4) buffer, as changing liquid buffer, crosses post 3 times, removes free acridinium ester and byproduct of reaction, obtain acridine-
KLH solution.
EDC (final concentration of 10mmol/L) and NHS is added (final concentration of in the acridine-KLH solution that above-mentioned purification is crossed
20mmol/L).After 25 DEG C are reacted 10 minutes, add TSH antibody (producer: Santa cruz biotechnology, article No.: sc-
418393), mixing, be placed in 25 DEG C reaction 4h, with the desalting column (Thermofish company) of 5mL 7KD molecular cut off with
150mM PBS (pH 7.4) buffer, as changing liquid buffer, crosses post 3 times, removes free EDC, NHS and byproduct of reaction,
Obtain the TSH monoclonal antibody solution of the band KLH of acridinium ester label.
Embodiment 6
Being dissolved in by 1mg KLH in 1mL 150mM PBS (pH 7.4), (10mg/mL is molten to add 5 μ L acridinium esters
Solution is in DMF) react 4h in 25 DEG C, with the desalting column (Thermofish company) of 5mL 7KD molecular cut off with 150mM PBS
(pH 7.4) buffer, as changing liquid buffer, crosses post 3 times, removes free acridinium ester and byproduct of reaction, obtain acridine-
KLH solution.
EDC (final concentration of 10mmol/L) and NHS is added (final concentration of in the acridine-KLH solution that above-mentioned purification is crossed
20mmol/L).After 25 DEG C are reacted 10 minutes, add estradiol antigen (producer: abcam, article No.: ab120657), mixing, be placed in
25 DEG C of reaction 4h, buffer with 150mM PBS (pH 7.4) with the desalting column (Thermofish company) of 5mL 7KD molecular cut off
Liquid, as changing liquid buffer, crosses post 3 times, removes free EDC, NHS and byproduct of reaction, obtains the band KLH of acridinium ester label
Estradiol antigenic solution.
Comparative example 1
1mg TSH antibody (producer: Santa cruz biotechnology, article No.: sc-418393) is dissolved in
In 1mL150mM PBS (pH 7.4), EDC (final concentration of 10mmol/L) and NHS (final concentration of 20mmol/L), 25
DEG C reaction 10 minutes after, add 16 μ L acridinium ester (10mg/mL is dissolved in DMF), mix in 25 DEG C react 4h, cut with 5mL 7KD
Stay the desalting column (Thermofish company) of molecular weight using 150mM PBS (pH 7.4) buffer as changing liquid buffer, cross post 3
Time, remove EDC, NHS and the byproduct of reaction of free acridinium ester, obtain the TSH monoclonal antibody solution of acridinium ester label.
Comparative example 2
1mg estradiol antigen (producer: abcam, article No.: ab120657) is dissolved in 1mL150mM PBS (pH
7.4) in, EDC (final concentration of 10mmol/L) and NHS (final concentration of 20mmol/L), after 25 DEG C are reacted 10 minutes, add 40 μ L
Acridinium ester (10mg/mL is dissolved in DMF), mixes and reacts 4h in 25 DEG C, with the desalting column of 5mL 7KD molecular cut off
(Thermofish company), using 150mM PBS (pH 7.4) buffer as changing liquid buffer, crosses post 3 times, removes free acridine
EDC, NHS of ester and byproduct of reaction, obtain the estradiol antigenic solution of acridinium ester label.
Test case
Anti-tsh antibody-solutions and contrast to the acridinium ester label band carrier protein obtained in embodiment 1,3,5 respectively
The anti-tsh antibody-solutions of the acridinium ester label that example 1 obtains with carbodlimide method detects for chemiluminescence immune assay.
In the TSH sample of 20 μ IU/mL, add the 20 μ g coated magnetic beads of TSH monoclonal antibody, then be separately added into each side
40ng/mL acridinium ester label anti-tsh antibody prepared by method, is placed in Full-automatic chemiluminescence immunoassay analysis meter (Asia, Shenzhen brightness dragon
Biotechnology Ltd., model: iFlash 3000) measure luminous value, parallel carry out 3 points of measurement, office's value of making even, tie
Fruit is shown in Table 1.The luminous value of immunoassay acquired results is the biggest, illustrates that the activity of acridine label is the best.
Table 1: the acridine markers tests TSH comparing result of each method labelling
As seen from the results in Table 1, the acridine label reagent of embodiment 1,3,5 preparation, the activity of reagent is significantly better than contrast
The acridine label reagent of example 1 preparation.
The goat polyclonal antibodies solution of the anti-tsh of the acridine labelling obtained in embodiment 1 is used for the TSH of series concentration
Chemiluminescence immune assay detection.20ug TSH monoclonal antibody bag it is separately added in the TSH sample solution of series concentration
The magnetic bead reagent of quilt and be separately added into 40ng/mL acridinium ester label anti-tsh antibody prepared by each method again, is placed in full-automatic chemical
Luminous value measured by luminescence immunoassay instrument (Ya Huilong Biotechnology Ltd. of Shenzhen, model: iFlash 3000),
Parallel carry out 3 points of measurements, office's value of making even, the results are shown in Table 2.With TSH antigen concentration as X-axis, with relative light unit as Y-axis, by table 2
Data are mapped, and obtain Fig. 2.
Table 2: the immune detection result of series concentration TSH antigen sample
| TSH antigen concentration (μ IU/mL) | Average irradiance (RLU) |
| 0.02 | 698 |
| 0.18 | 2967 |
| 0.39 | 4847 |
| 2.28 | 25098 |
| 11.55 | 92288 |
| 48.44 | 403477 |
| 86.48 | 702871 |
From table 2 and Fig. 2 result, the acridine label reagent of embodiment 1 preparation can be applicable to chemiluminescence immunoassay and divides
Analyse and respond well.The acridinium ester label preparation method of embodiment 1 has the good suitability.
By the estradiol antigenic solution of acridinium ester label band carrier protein obtained in embodiment 2,4,6 and comparative example 2
The estradiol antigenic solution of the acridinium ester label obtained with carbodlimide method detects for chemiluminescence immune assay.To 500pg/
In the estradiol sample of mL, add the 40 μ g coated magnetic beads of estradiol monoclonal antibody, then be separately added into prepared by each method
40ng/mL acridinium ester label estradiol antigenic solution, (Asia, Shenzhen brightness dragon is biological to be placed in Full-automatic chemiluminescence immunoassay analysis meter
Technical concern company limited, model: iFlash 3000) measure luminous value, parallel carry out 3 points of measurements, office's value of making even, result is shown in
Table 1.The luminous value of immunoassay acquired results is the least, illustrates that the activity of acridine label is the best.
Table 3: the acridine markers tests E2 comparing result of each method labelling
| Acridine label | Average irradiance (RLU) |
| Embodiment 2 | 94734 |
| Embodiment 4 | 103267 |
| Embodiment 6 | 98247 |
| Comparative example 2 | 643026 |
As seen from the results in Table 3, the acridine label reagent of embodiment 2,4,6 preparation, the activity of reagent is significantly better than contrast
Example 2, the acridine label reagent that comparative example 2 prepares is almost without signal.
The estradiol antigenic solution of the band carrier protein of the acridine labelling obtained in embodiment 2 is used for the E2 of series concentration
Chemiluminescence immune assay detection.40ug estradiol monoclonal antibody bag it is separately added in the E2 sample solution of series concentration
The magnetic bead reagent of quilt and be separately added into 40ng/mL acridinium ester label anti-tsh antibody prepared by each method again, is placed in full-automatic chemical
Luminous value measured by luminescence immunoassay instrument (Ya Huilong Biotechnology Ltd. of Shenzhen, model: iFlash 3000),
Parallel carry out 3 points of measurements, office's value of making even, the results are shown in Table 4.In sample, E2 antigen concentration is as X-axis, with relative light unit as Y-axis,
Mapped by table 4 data, obtain Fig. 3.
Table 4: the immune detection result of series concentration E2 antigen sample
From table 4 and Fig. 3 result, the acridine label reagent of embodiment 2 preparation can be applicable to chemiluminescence immunoassay and divides
Analyse and respond well.The acridinium ester label preparation method of embodiment 2 has the good suitability.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. an acridine labelling conjugate, it is characterised in that the acridine substituent that includes being sequentially connected with, carrier protein and to be marked
Albumen；
Described carrier protein is containing carboxyl and the albumen of amino, modified protein, polypeptide or modified polypeptides, and described carrier protein leads to
Cross the amino on described carrier protein react with described acridine substituent formation chemical bond be connected；
Described albumen to be marked is the albumen containing amino, modified protein, polypeptide or modified polypeptides, on described albumen to be marked
Amino forms-NH-CO-structure thus by described carrier protein and described egg to be marked with the carboxyl reaction on described carrier protein
Connect together in vain.
Acridine labelling conjugate the most according to claim 1, it is characterised in that described acridine substituent is acridinium ester, a word used for translation
Pyridine acid, acridinium carboxamide or acridine sulfonamide.
Acridine labelling conjugate the most according to claim 1, it is characterised in that described carrier protein is bovine serum albumin
In vain, chicken serum albumin or hemocyanin.
4. according to the acridine labelling conjugate described in claim 1 or 3, it is characterised in that described albumen to be marked be antigen, half
Antigen or antibody.
5. the preparation method of the acridine labelling conjugate according to any one of a Claims 1 to 4, it is characterised in that include
Following steps:
Acridine substituent-carrier protein conjugate is obtained after acridine substituent and carrier protein covalent cross-linking, fully reaction, its
In, described carrier protein is containing carboxyl and the albumen of amino, modified protein, polypeptide or modified polypeptides, and described carrier protein leads to
Cross the amino on described carrier protein react with described acridine substituent formation chemical bond be connected；
Described acridine substituent-carrier protein conjugate is purified；
With cross-linking agent, the carboxyl on described acridine substituent-carrier protein conjugate after purification is activated；And
Obtain after the described acridine substituent-carrier protein conjugate after activated carboxylic and protein-crosslinking to be marked, fully reaction
To acridine labelling conjugate, wherein, described acridine labelling conjugate includes the acridine substituent, the carrier protein that are sequentially connected with and treats
Labelled protein, described albumen to be marked is the albumen containing amino, modified protein, polypeptide or modified polypeptides, described egg to be marked
Amino on Bai forms-NH-CO-structure with the carboxyl reaction on described carrier protein thus by described carrier protein with described treat
Labelled protein connects together.
The preparation method of acridine labelling conjugate the most according to claim 5, it is characterised in that described acridine substituent
In operation with carrier protein covalent cross-linking, the mol ratio of described acridine substituent and described carrier protein is 100～20000:
1。
The preparation method of acridine labelling conjugate the most according to claim 6, it is characterised in that after described activated carboxylic
Described acridine substituent-carrier protein conjugate and protein-crosslinking to be marked operation in, described acridine substituent-carrier egg
White conjugate is 5:1～1:5 with the mol ratio of described albumen to be marked.
The preparation method of acridine labelling conjugate the most according to claim 5, it is characterised in that described cross-linking agent is to pure
The carboxyl on described acridine substituent-carrier protein conjugate after change carries out in the operation activated, and described cross-linking agent includes carbon
Diimine and N-Hydroxysuccinimide.
The preparation method of acridine labelling conjugate the most according to claim 8, it is characterised in that described carbodiimide is selected from
In dicyclohexylcarbodiimide, 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and N, N'-DIC
At least one, described carbodiimide is 10～5000:1 with the mol ratio of described acridine substituent-carrier protein conjugate；
At least one in N-hydroxy-succinamide and NHS of described N-Hydroxysuccinimide,
Described carbodiimide is 5:1～1:10 with the mol ratio of described N-Hydroxysuccinimide.
10. a chemical luminescence reagent kit, is formed as according to any one of Claims 1 to 4 for combining albumen to be marked
Acridine labelling conjugate, it is characterised in that described chemical luminescence reagent kit includes: acridine substituent and carrier protein；
Described carrier protein is that described carrier protein can containing carboxyl and the albumen of amino, modified protein, polypeptide or modified polypeptides
It is connected reacting formation chemical bond with described acridine substituent by the amino on described carrier protein；
Described albumen to be marked is the albumen containing amino, modified protein, polypeptide or modified polypeptides, on described albumen to be marked
Amino can form-NH-CO-structure with the carboxyl reaction on described carrier protein thus described carrier protein and described waiting be marked
Note albumen connects together.
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| CN112684163A (en) * | 2021-01-29 | 2021-04-20 | 安邦（厦门）生物科技有限公司 | Acridine compound marking raw material working solution and preparation method thereof |
| CN114113610A (en) * | 2021-12-08 | 2022-03-01 | 深圳市亚辉龙生物科技股份有限公司 | Acridinium ester labeled compound and detection kit |
| CN117986328A (en) * | 2024-04-03 | 2024-05-07 | 江西省转化医学研究院 | Cyclic peptide luminescent coupling molecule and preparation method and application thereof |
| CN118033151A (en) * | 2024-04-11 | 2024-05-14 | 江西省转化医学研究院 | Homogeneous chemiluminescent procalcitonin detection reagent constructed by cyclopeptide luminescent coupling molecules |
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